The Clinical Significance Of RSK2 Expressed In Osteosarcomes And The Effects Of RSK2 On Osteosarcome Cell Lines

Objective:To investigate the expression and clinical significance of p90 ribsomal S6 kinase 2(RSK2) in osteosarcomes(OS), to construct eukaryotic expression vector eneoding RNA interference sequences specific for RSK2 gene, to observe its effect on osteosarcome cell lines(MG-63,143 B,U2-OS), and to study the molecular mechanism of RSK2 on osteosarcome cell lines.Methods:1. Immunohistochemical staining was performed to determine RSK2 expression in 30 cases of osteosarcomes and 15 cases of non-neoplastic normal tissues away from the tumors. Clinical history of all the cases were collected and analyzed.2. The shRNAs sequences based on the sequence of RSK2 messenger RNA(mRNA) in the GenBank were designed to silence the expression of RSK2 gene and one scrambled shRNA sequence as negative control.Reverse transcription polymerase chain reaction(RT-PCR) assay and western blot experiment were used to detect the effect of RSK2 shRNA onthree OS cell lines. Flow cytometry was utilized to detect the impact of silencing of RSK2 on the cell cycle and apoptosis of osteosarcome cells.Cell Counting Kit-8 assays(CCK8) was used to analysis the cell proliferation and drug chemosensitivity. Xenograft tumor nude mice model was built to evaluate the ability of tumorigenesis. Transwell migration assays were used to determine the ability of the migration.3. Western blot assay was performed to determine the expression of ERK, phosphorylated ERK(p-ERK), AKT, phosphorylated AKT(p-AKT),mTOR, phosphorylated mTOR(p-mTOR), c-Fos, Bcl2, Bax, and caspase3 in OS cell lines after transfected with RSK2 shRNA.Results:1. The frequency of RSK2 expression in osteosarcome and normal tissues was 86.67% 、 26.67%, respectively. RSK2 was more frequently(p<0.05) expressed in osteosarcome than in normal tissues but it was no difference in the tumors from women than men. RSK2 expression in osteosarcomes was also positively correlated to the tumor size and it was more frequently detected in the tumors with ≥3cm than in the tumors with<3cm, the frequence was 95% and 70% respectively. The RSK2 expression was correlated with the clinical staging. The frequency of stage I, II, III was50%, 95.45% and 100%, respectively, the difference between stage I and stage II, III was statistically significant(P<0.05). The RSK2 expression was also correlated with the pathological grading and the frequency ofgrade I, II, and III was 66.67%, 90.91%, 92.30%, respectively. The difference between stage I and stage II, III was statistically significant(P<0.05). The expression of RSK2 was associated with alkaline phosphatase(ALP) levels. The positive rate in the cases with evaluated ALP was 91.67%, and in the non-evaluated was 66.67%, the difference was statistically significant(P<0.05).2. The RSK2 shRNA we designed could effectively silence the expression of RSK2 mRNA and protein. After transfected with RSK2 shRNA, all three OS cell lines had an increased apoptosis, and the cells were mainly in the late stage of apoptosis. RSK2 shRNA could block the OS cell cycle in G0-G1 phase. Silencing of RSK2 also significantly decreased the proliferation of cells and the ability of tumorigenesis on xenograft tumor nude mice model. After transfected with RSK2 shRNA,the ability of migration decreased obviously and the chemosensitivity to cis-platinum and Doxorubicin elevated significantly.3. The expression of p-AKT, p-mTOR, c-Fos and Bcl2 were obviously inhibited by using shRNA to silence the RSK2 expression in the osteosarcome cells. In contrast, the level of Bax increased significantly.While the expression of ERK, p-ERK and caspase3 were no significant changes.Conclusion1. The frequency of RSK2 expression was much higher inosteosarcomes than in the normal tissues. The RSK2 expression was correlated to the tumor size, clinical staging, pathological grading of osteosarcome and the tumors with high levels of ALP tended to be more frequently positive for RSK2. There was no correlation of RSK2 expression with the sex and the age.2. The expression of RSK2 in osteosarcome cells was related to the cell cycle and apoptosis. Downregulated of RSK2 could decrease the proliferation and migration of osteosarcome cells. Inhibition of RSK2 activity was able to increase the chemosensitivity and tumorigenesis.3. Knockdown of RSK2 mediates the biological behavior of OS cells through inactivating the AKT/mTOR signal pathway and influence the occurrence and development of tumor through Bcl2, Bax and c-Fos.Silence of RSK2 could not activate the expression of ERK and caspase3.